[Frontiers in Bioscience 19, 1105-1116, June 1, 2014] 1105 Carboxylation-dependent conformational changes of human osteocalcin Andrea Cristiani 1 , Fabio Maset 2 , Luca De Toni 3 , Diego Guidolin 4 , Davide Sabbadin 5 , Giacomo Strapazzon 3,6 , Stefano Moro 5 , Vincenzo De Filippis 2 , Carlo Foresta 3 1 CRS4, Biomedicine sector, Parco Polaris, 09010 Pula (CA), Italy, 2 Laboratory of Protein Chemistry, Department of Pharmaceutical and Pharmacological Sciences, University of Padova, 35131 Padova, Italy, 3 Department of Molecular Medicine and Centre for Human Reproduction Pathology, University of Padova, 35121 Padova, Italy, 4 Department of Human Anatomy and Physiology, University of Padova, 35121 Padova, Italy, 5 Molecular Modeling Section (MMS), Department of Pharmaceutical and Pharmacological Sciences, University of Padova, 35131 Padova, Italy, 6 EURAC Institute of Mountain Emergency Medicine, 39100 Bozen/Bolzano, Italy TABLE OF CONTENTS 1. Abstract 2. Introduction 3. Materials and Methods 3.1. Analytical techniques 3.2. Spectroscopic techniques 3.3. Thermal stability measurements 3.4. Homology modeling 3.5. Molecular dynamics 4. Results 4.1. Effect of γ-carboxylation on the conformation and affinity of OCN for calcium 4.2. Effect of γ-carboxylation on the stability of OCN in the apo- and holo-form 4.3. Homology modeling of human OCN 4.4. Molecular dynamics simulations and calcium binding to Glu-OCN and Gla-OCN 4.4.1. Gla-OCN 4.4.2. Glu-OCN 4.5. Steered molecular dynamics 5. Discussion 6. Acknowledgements 7. References 1. ABSTRACT Osteocalcin (OCN) is a small noncollagenous protein mainly produced by osteoblasts and is highly represented in bones of most vertebrates. Human OCN contains up to three gamma-carboxyglutamic acid (Gla- OCN) residues at positions 17, 21 and 24 which are thought to increase calcium binding strength, improving mechanical properties of the bone matrix. Recent studies revealed that OCN exerts also important endocrine functions, affecting energy metabolism and male fertility. The latter effect seems to be mediated by the uncarboxylated form of OCN (Glu-OCN). We employed human and mouse OCN as models of fully carboxylated and uncarboxylated OCN forms to investigate, by the use of circular dichroism and molecular dynamics simulations, the respective conformational properties and Ca 2+ affinity. Ca 2+ binding was found to trigger a similar conformational transition in both Glu-OCN and Gla-OCN, from a disordered structure to a more compact/stable form. Notably, gamma- carboxylation increases the affinity of OCN for Ca 2+ by > 30 fold suggesting that, in physiological conditions, Gla- OCN is essentially Ca 2+ -bound, whereas Glu-OCN circulates mainly in the Ca 2+ -free form. 2. INTRODUCTION Osteocalcin (OCN) is a small protein of 46-50 amino acid residues, mainly produced and secreted by osteoblasts. It represents one of the most abundant (10-20%) noncollagenous protein in the bone tissue of most vertebrates examined to date, from bony fish to mammals, and its primary structure is highly conserved among vertebrates (1). In particular, human and porcine OCN contain up to three ?-carboxyglutamic acid (Gla) residues at positions 17, 21 and 24 (2). However, the glutamic acid at position 17 (Glu17) is ?-carboxylated only in about 9% of human OCN molecules (3). Carboxylation of Glu-residues is a vitamin K dependent post-translational modification that is thought to increase affinity of OCN for calcium ions (Ca 2+ ) and bone hydroxyapatite, thus contributing to bone formation (4). Circular dichroism and nuclear magnetic resonance analyses indicate that fully γ- carboxylated chicken and bovine osteocalcin (Gla-OCN) in solution are largely unstructured in the absence of calcium (apo-OCN) and that, only after addition of physiological Ca 2+ concentrations (1.0-1.3 mM), they undergo a transition to a folded state (holo-OCN) displaying highly flexible N- and C-terminal regions characterized by a protein core formed by three α-helical segments stabilized